The specific mechanisms that control differentiation and maturation of goblet cells from intestinal stem cells have not yet been defined. Understanding how goblet cell specification occurs is important because without this knowledge, therapeutic interventions designed to control cellular composition of the intestinal epithelium will remain difficult to achieve. Our long term goal is to define the mechanism of differentiation of all cell types within the intestinal epithelium, as a means to develop novel therapeutic strategies based on controlled production of specific cell types. The objective of this R03 application is to identify the key components of the transcriptional cascade that controls differentiation of goblet cells, and to develop additional preliminary data for an R01 application to pursue this novel line of investigation. The central hypothesis of this application is that the SAM pointed domain-containing ETS transcription factor (Spdef, also called Pdef or PSE) directs differentiation of goblet cells from fate-restricted secretory progenitors specified by Atoh1 and Gfi1.Two specific aims will test the central hypothesis: 1) Define the requirement and sufficiency for Spdef to direct goblet cell differentiation. 2) Position Spdef within the transcriptional network that controls differentiation of intestinal cells. In Aim 1, our working hypothesis is that Spdef is both necessary and sufficient to direct differentiation of bipotential goblet/Paneth progenitor into goblet cells. We will define the effect of manipulating Spdef levels in a novel colon cancer cell model of differentiation and in transgenic mice by assessing changes in cellular composition, homeostasis, and protein/gene expression changes. The hypothesis for Aim 2 is that a transcriptional hierarchy controls the fate of progressively more restricted progenitors to achieve differentiation of individual secretory cell types. Specifically, we hypothesize that for goblet cells, Hes1 represses Atoh1;in the absence of Hes1, Atoh1 activates Gfi1 expression, which regulates expression of Spdef to drive goblet cell differentiation (schematized in Figure 1, Specific Aims page). Here we will identify the mechanisms by which Hes1, Atoh1, and Gfi1 control Spdef expression. Upon completing these studies, we expect to have demonstrated the essential roles for Spdef as a key factor in goblet cell differentiation, and defined the mechanistic framework for transcriptional control of Spdef. The proposed research is innovative in defining a novel gene that controls goblet cell production. Furthermore, these results will contribute to the broader understanding of how distinct epithelial cell types arise from multipotent progenitors throughout the body. With the additional data provided by these studies, the applicant will be well-positioned to obtain R01 funding to investigate the role of Spdef in normal and abnormal function of the intestine. PUBLIC HEALTH RELEVANCE: This project has relevance for the NIH's mission to reduce the burdens of illness and disability, as a component of research into the causes, prevention, and cure of human diseases, and the processes of human growth and development. The proposed studies are important to public health because the mechanisms identified that control goblet cell differentiation will provide targets to control pathologic changes to the intestine in diseases such as colorectal cancer and inflammatory bowel disease. Once these methods are available, it will be possible to produce or reduce specific cell types on demand.